The long-term goal of this proposal is to develop a safe, effective and user-friendly vitrification system for cryopreservation of whole human ovaries. In the U.S., over 140,000 girls and women age 0-49 are diagnosed with cancer annually. While improved diagnosis and treatment have led to increased survival rates (75% in premenopausal and 85% in childhood cancer patients), devastating side effects of cancer therapies include premature ovarian failure, infertility and menopause-related health risks. While cryopreservation of embryos and oocytes are effective and will remain as the most commonly used fertility preservation method, ovarian cortex and whole ovary cryopreservation is the only option for future fertility in prepubertal patients and those who require immediate cancer treatment. Cryopreservation of whole ovaries, compared to ovarian tissue, has added advantages of 1) preserving all classes of follicles, which can be used for in vitro maturation, 2) longer graft lifespan, and 3) faster recovery supported by existing blood vessels after transplantation with vascular anastomosis. However, current methods for whole ovary cryopreservation are suboptimal and there is no live birth in human from cryopreserved whole ovaries, despite it may be the best choice for prepubertal cancer patients for future fertility and intact endocrine function for decades without the help of exogenous hormones. The proposed research is part of 21st Century Medicine?s more general program of developing superior cryopreservation system, including cryoprotective agent (CPA) solutions, perfusion apparatus (hardware and software) for CPA loading and unloading, and liquid nitrogen storage units, for organs. Our company has successfully demonstrated functional preservation of vitrified rabbit kidneys after transplantation. The pig and human ovaries are similar in size and smaller (~3-5 grams), compared to rabbit kidneys (15-20 grams), which makes vitrification of pig and human ovaries more achievable than rabbit kidneys. In this SBIR Phase I study, we will establish optimal perfusion parameters and CPA combination that will allow the preservation of follicles and blood vessels in whole swine ovaries, and design an ovary-specific perfusion apparatus (Aim 1). We will also optimize cooling and warming rates, and determine whether radiofrequency heating can be utilized to improve efficiency and uniformity during rewarming of vitrified swine ovaries (Aim 2). Ovarian function will be evaluated in vitro by ovarian tissue culture (histology and immunohistochemistry), preantral follicle culture (survival, antrum formation, hormone production), cumulus-oocyte-complex culture (oocyte maturation). Results from these studies will improve current technology in whole ovary cryopreservation and include a design and prototype of an automated vitrification system. Future Phase II studies will focus on construction of the vitrification apparatus incorporating aspects of perfusion, cooling, storage and rewarming with a user- friendly computer interface. This novel system will be tested in vivo (to demonstrate livebirth in the swine model after transplantation) and in vitro (to test ovarian function using human ovaries) in Phase II.